Circulating acetaminophen metabolites are toxicokinetic biomarkers of acute liver injury ADB Vliegenthart 1 , RA Kimmitt 1 , JH Seymour 1 , NZ Homer 1 , J I Clarke 2 , M Eddleston 1 , A Gray 3 , D M Wood 4,5 , P I Dargan 4,5 , JG Cooper 6 , D J Antoine 2 , DJ Webb 1 , SC Lewis 7 , DN Bateman 1 & JW Dear 1 . Affiliations: 1 Pharmacology, Toxicology and Therapeutics, University/BHF Centre for Cardiovascular Science, University of Edinburgh, UK. 2 MRC Centre for Drug Safety Science, Department of Molecular & Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. 3 Emergency Medicine Research Group, Department of Emergency Medicine, Royal Infirmary of Edinburgh, Edinburgh, UK. 4 Clinical Toxicology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK. 5 King’s College London, London, UK. 1
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Circulating acetaminophen metabolites are toxicokinetic biomarkers of
acute liver injury
ADB Vliegenthart1, RA Kimmitt1, JH Seymour1, NZ Homer1, J I Clarke2, M
Eddleston1, A Gray3, D M Wood4,5, P I Dargan4,5, JG Cooper6, D J Antoine2, DJ Webb1,
SC Lewis7, DN Bateman1 & JW Dear1.
Affiliations:
1Pharmacology, Toxicology and Therapeutics, University/BHF Centre for
Cardiovascular Science, University of Edinburgh, UK.
2MRC Centre for Drug Safety Science, Department of Molecular & Clinical
Pharmacology, Institute of Translational Medicine, University of Liverpool,
Liverpool, UK.
3Emergency Medicine Research Group, Department of Emergency Medicine,
Royal Infirmary of Edinburgh, Edinburgh, UK.
4Clinical Toxicology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK.
5King’s College London, London, UK.
6Emergency Department, Aberdeen Royal Infirmary, Aberdeen, UK.
7Centre for Population Health Sciences, University of Edinburgh, Edinburgh, UK.
Contact Information: Dr James Dear PhD FRCPEdin, Edinburgh University/BHF
Centre for Cardiovascular Science, Queen's Medical Research Institute, 47 Little
France Crescent, Edinburgh, EH16 4TJ. UK, Tel 44 131 2429215, Email:
monitored for were m/z 152 110.0, 93.1 at 20 and 13 V and m/z 156.1
114.1, 97.1 at 15 and 22 V for APAP and APAP-d4, respectively. For the positively
ionised APAP metabolites; APAP-Cys, APAP-Mer and APAP-GSH, m/z 271.1
182.0, 207.6 at 8 and 9 V, m/z 313.0 140.1, 208.1 at 28 and 16 V and m/z
457.2 140 at 33 V were monitored for.
For the negatively ionised APAP metabolites APAP-Sul, APAP-Glu and the
internal standard APAP-SUL-d3 m/z 229.8 107.0, 150.1 at 36 and 15 V, m/z
326.0 113.0, 150.0 at 28 and 16 V and m/z 233.0 109.5, 181.4 at 30 and 5 V
were monitored for.
Statistical analysis
All data are presented as median and interquartile range (IQR), except for
receiver operator characteristic (ROC) data where 95% confidence intervals are
quoted. Comparisons were made using the Mann-Whitney U test. All LC/MS-MS
data were transformed from mass to molar concentrations before analyses were
performed. APAP plasma half-life was estimated using non-linear fit, assuming
first order kinetics. All calculations and ROC analysis were performed using
GraphPad Prism software (GraphPad Software, La Jolla, CA). Logistic regression
models were run using SAS version 9.4.
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Study Highlights
What is the current knowledge on the topic?
Acetaminophen overdose is common. Decisions regarding need for treatment are
frequently based on measurement of the blood acetaminophen concentration.
However, acetaminophen must be metabolised to cause liver injury.
Acetaminophen metabolites are present in the circulation after therapeutic
dosing and overdose.
What question did this study address?
Are circulating acetaminophen metabolites elevated with acute liver injury and
can they predict injury better than acetaminophen parent drug concentration?
What this study adds to our knowledge
Patients who developed acute liver injury had higher acetaminophen metabolites
derived from the cytochrome P450 pathway that mediates toxicity. Hospital
presentation metabolites were more sensitive and specific for liver injury
compared with the parent drug.
How this might change clinical pharmacology or translational science
Acetaminophen metabolites can predict liver injury and have potential utility in
stratified trials and for refinement of clinical decision-making.
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Acknowledgements Author ADBV was supported by an NC3Rs PhD Studentship
(NC/K001485/1). Author JWD acknowledges the support of NHS Research
Scotland (NRS) through NHS Lothian and a BHF Centre of Research Excellence
Award.
Conflict of Interest/Disclosure: The authors have no conflicts of interest to
disclose.
Author Contributions: JWD and ADBV wrote manuscript, ADBV, RAK, JHS, NZH,
JIC performed research, ME, AG, DMW, PID and JGC recruited patients, JWD, DJA,
DJW and DNB designed and reviewed research and SCL performed statistical
analysis.
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Table and Figure Legends
Table 1. Patient characteristics of the discovery cohort divided by absence or
presence of acute liver injury †according to the British National Formulary 2009
(37). P-value for difference between groups was determined by Mann Whitney
test or Chi-square test.
Table 2 Patient characteristics of the validation cohort divided by absence or
presence of acute liver injury (>50% increase in ALT). P-value for difference
between groups was determined by Mann Whitney test or Chi-square test.
Table 3. Predictive accuracy of current and new biomarkers. Table with ROC-
AUC (area under the curve with 95% CI), sensitivity (at 90% specificity) with
95% CI, and statistical significance for different metabolites measured at pre-
treatment in the discovery and hospital presentation in the validation cohort. P-
value represents significance level compared to ROC-AUC = 0.5. The positive and
negative predictive values (PPV and NPV, respectively) are also presented for
each metabolite/biomarker.
Table 4. Effect of APAP-Cys/APAP-Sul on acute liver injury in patients treated
with ondansetron compared to placebo in SNAP trial. *Adjusted by the variables
in the minimisation algorithm, and centre. † Obtained with a model in which only
treatment and regimen were included.
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Figure 1. Pathways of acetaminophen (APAP) metabolism. APAP-sulphate